Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.Over the last 2 decades, the development of molecular biology tools has led to the emergence of a new discipline, molecular microbial ecology. The overall structural diversity of microbial communities can be examined easily using PCR-based strategies (6), usually targeting the 16S rRNA gene as a universal genetic marker of prokaryotes. Genotyping approaches avoid current limitations of cultivation methods, which only poorly reflect the phylogenetic diversity of microbial communities (12). The principles, technical aspects, and limitations of commonly employed methods were recently reviewed (10). Among these methods, terminal restriction fragment length polymorphism (T-RFLP) has proved to be invaluable for rapid characterization of the composition and dynamics of species-rich samples (13). Compared to other approaches, T-RFLP is semiquantitative and combines high levels of sensitivity, resolution, and reproducibility (see Table S1 in the supplemental material). Taxonomic diversity of microbial communities is evaluated by using the strain-dependent variability of restriction sites within a conserved PCR-amplified DNA fragment. The terminal restriction fragments (T-RFs) of digested PCR products appear as chromatographic peaks after size-dependent electrophoretic separation due to a fluorescent tag attached to one of the primers used for PCR. The relative abundance of peaks is evaluated, and fragment lengths are estimated using a fluorescent internal size standard comigrating with the sample (5). The estimated lengths corresponding to the T-RFLP peaks obtained are compared to databases of T-RF sizes generated by in silico digestion of known 16S rRNA gene sequences with commonly used restriction enzymes for phylogenetic assignment (13). However, estimation of T-RF lengths from experimental chromatograms is biased by the fact that differences in the electrophoretic properties of the two different fluorescent dyes used to distinguish sample fragments from the size marker significantly affect fragment migration (7, 11). Discrepancies greater than 6 nucleotides (nt), depending on the length of the fragment, have been reported between expected and experimentally estimated fragment lengths (7). This causes errors in phylogenetic assignments and may in turn lead to erroneous inferences regarding the functional aspects of the microbial communities under investigation. Another drawback of T-RFLP is the difficulty of retrieving sequence information directly from experimental T-RFs, since additional construction of representative 16S rRNA gene libraries is required to obtain such information.Here we propose an experimental strategy to circumvent current limitations of T-RFLP and facilitate characterization of microbial communities. First, we propose an optimized protocol for T-RFLP that yields reliable T-RF sizes. Second, we describe use of denaturing high-performance liquid chromatography (D-HPLC) as an alternative to cloning in order to gain direct access to DNA sequence information. D-HPLC, an emerging technique for microbial community profiling (1, 4), enables collection of DNA fragments separated on the basis of differences in sequence, sequence length, and G+C content at a partially denaturing temperature. The unambiguous phylogenetic characterization of a model microbial assembly of five reference strains is described as proof of principle of the usefulness of the proposed strategy.